1. Field of the Invention
The present invention in the field of biology provides a novel method and culture system for long term culture of functional hepatocytes by first preparing hepatocyte spheroids and inoculating these into a specialized bioreactor that is a rotating wall vessel wherein the cells are cultured under conditions that promote three-dimensional aggregation and cellular differentiation.
2. Description of the Background Art
Hepatocyte Spheroids
Landry, J. et al., J. Cell Biol. 101: 914-923 (1985) described the spontaneous formation of spheroidal aggregates when primary rat liver cells were incubated on a nonadherent plastic dish. Three distinct cell morphologies were noted: surface monolayer cells; hepatocytes grouped as islands with deposition of extracellular matrix components; and structures resembling bile ducts. Tong, J Z, et al., Exp. Cell Res. 189:87-92 (1990), studied multicellular spheroids formed from newborn rat liver cells and detected secretion of liver proteins, specifically albumin and transferrin, for up to 60 days when the culture medium was supplemented with dexamethasone, glucagon, insulin, and epidermal growth factor. These investigators (Tong, J Z, et al., Exp. Cell Res. 200:326-332 (1992)) also observed maintenance of liver-specific functions in spheroid cultures of adult rat hepatocytes, demonstrating metabolism of lidocaine by the cytochrome P450 (CYP) enzyme 3A2, for up to 14 days. CYP 1A1 was strongly induced by methylcholanthrene, remaining constant for up to 22 days.
Interestingly, the presence of serum factors inhibited spheroid formation under certain conditions (Koide, N. et al., Exp. Cell Res. 186:227-235 (1990)).
One of the present inventors and his colleagues (A. P. Li et al., In Vitro Cell. Dev. Biol. 28A:673-677 (1992a)) was the first to report spheroid formation by human hepatocytes. Their simple, yet proven, method involved seeding 5×106 hepatocytes in culture medium on a 100-mm plastic dish and shaking at 50 rpm overnight. This method caused 90% of the cells to aggregate in the form of spheroids, which were shown to possess many of the morphological characteristics of intact liver.
U.S. Pat. No. 5,624,839 disclosed that lipid-bound glycosaminoglycan promoted spheroid formation.
Cytochrome P450s (CYPs)
CYPs are a family of enzymes, localized to the cytoplasmic side of the endoplasmic reticulum of the liver cell, that catalyze the oxidation of organic compounds, resulting in increased water solubility which promotes excretion from the cell. CYPs are obviously important for processing xenobiotics. Table 2 lists a number of CYP enzymes in rat liver that are responsible for metabolism (and detoxification) of a number of drugs.
Once hepatocytes are isolated from the liver and are grown in conventional primary cultures, the activity of these important enzymes is rapidly lost. This loss is particularly prominent for rat hepatocytes which lose 80% of their CYP activity in the first 24 hours of culture (Paine, A J, In: Berry, Minn. et al. (eds.), The Hepatocyte Review, Kluwer Academic Publishers, Netherlands, pp. 411-420, 2000).
Rotating Wall Culture Vessels
Rotating wall vessels or RWVs are a class of bioreactors developed by and for NASA beginning in about 1990 that were designed to grow suspension cultures of animal cells in a quiescent environment that simulates microgravity. RWVs were first described in a number of U.S. patents (U.S. Pat. Nos. 5,026,650; 5,153,131; 5,153,133) assigned to NASA, and thereafter in several additional patents (U.S. Pat. Nos. 5,437,998; 5,665,594; 5,702,941) assigned to Synthecon, Inc., who served as a contractor and licensee of NASA Other patents describe the same principle as the RWV, i.e., horizontal rotation for mixing or suspending cells in culture medium. With the exception of Ingram et al. (U.S. Pat. No. 5,523,228), however, these patents do not disclose the culture of freely suspended cells. For example, in Rhodes et al. (U.S. Pat. No. 5,104,802), cells are confined inside a hollow fiber rotating with the culture vessel. U.S. Pat. No. 6,117,674 described a process for propagating a pathogen in a three-dimensional tissue mass in RWV culture. The foregoing patents are all incorporated by reference in their entirety.
During operation, an RWV is completely filled with medium and rotates about a horizontal axis. Oxygenation occurs in a bubble-free manner via a silicone rubber membrane that covers the back wall of the cultivation chamber. Cells are evenly distributed and semi-buoyant during cultivation, and mixing is accomplished without stirring by end-over-end rotation of the vessel (Schwarz, R P, et al., J. Tiss. Cult. Meth. 14:51-58, 1992; Cowger, N L, et al., Biotechnol. Bioeng 64:14-26, 1999).
RWVs have proven beneficial to the cultivation of many cell types for tissue engineering applications. Unlike conventional vessels, a RWV accommodates three-dimensional (3D) assembly and co-location of dissimilar cell types in a gently mixed environment. The result is more extensive 3D growth with increased cell-cell and cell-matrix interactions and cellular differentiation that more closely resembles organized living tissue (Spaulding, G F, et al., J. Cell. Biochem. 51:249-251, 1993). These properties of RWVs have been exploited to grow and study primary cells from various normal tissues (Goodwin, T J, et al., Proc. Soc. Exp. Biol. Med. 202:181-192, 1993; Freed, LE et al., In Vitro Cell. Dev. Biol. 33:381-385, 1997) and cells from tumors. For example, aggregates of human prostate tumor cells were more differentiated in terms of their growth, morphology, and cytoskeletal protein expression when cultured in a RWV compared to “control” tumor cells grown in conventional spinner flasks or static cultures (Clejan, S. et al, Biotechnol. Bioeng. 50:587-597, 1996). Khaoustov, VI, et al., In Vitro Cell. Dev. Biol. 35:501-509. 1999) described the culture of human hepatocytes in an RWV, primarily providing morphological descriptions, though this document disclosed continuous albumin secretion and urea nitrogen production over a period of 20 days.
Of the existing patent disclosures describing functional hepatocytes in vitro, with utility as “artificial livers,” none describe or suggest the use of freely suspended cells in a rotating bioreactor. For example, Li et al. (U.S. Pat. No. 5,270,192) disclosed a hepatocyte bioreactor in which hepatocytes or aggregates are entrapped inside a matrix of glass beads. In connection with hepatocyte spheroid formation, U.S. Pat. No. 5,624,839 (noted above) disclosed a composition that promoted this process.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.